DESCRIPTION: The broad, long term objective of this application is to use NMR spectroscopy to investigate the structures of respiratory proteins; in particular, how O2 and CO bind to myoglobin and hemoglobin as well as model systems, such as "picket fence" and "capped" or protected metalloporphyrins. The health relatedness of this work is that a full understanding of how O2 vs. CO binding is controlled by proteins is important for understanding how O2 is transported in blood and muscle, how O2 affinities are changed in various respiratory diseases, as well as how in the long term, blood substitutes may be designed. The specific aims are first, to construct a multiple probe 600 MHz widebore NMR spectrometer, which will permit routine observations to be made on at least two different samples, simultaneously, in the same magnet. This will increase sample throughput, reducing the overall effective cost of expensive NMR resources. Second, CO bonding will be investigated in heme model systems, using 13C and 17O chemical shift and 17O quadruple coupling information and density functional theory (DFT). The goal here is to relate observed spectroscopic parameters to structure (ligand tilt, bend, electrostatic fields, heme ruffling/doming/saddling, proximal side interactions). The effects of electron correlation and exchange will be handled using the DFT formalism. The third aim is to clarify the nature of FeCO/distal/proximal interactions in heme proteins themselves. Particular emphasis will be placed on obtaining first principles, quantitative analyses of 13C, 17O shielding and field gradient tensors in A0, A1, and A3 substrates of myoglobins and hemoglobins. The fourth goal is to investigate how O2 binds to Fe in model heme systems, using methods developed for CO. The final goal is to study Fe-O2 interaction in heme proteins themselves, using 17O NMR and DFT. As with early reports for CO, reported FeOO geometries vary widely, from ~115 degrees to 159 degrees. However, it is likely that a much narrower range of angles is actually present, since the energies associated with these distortions are very large. Solid-state 17O NMR will enable a test of the hypothesis that the ligand geometries are much closer to 130 degrees. The extent of O2 stabilization will be investigated (e.g. H-bonding to the distal histidine), again using DFT methods. The goal is to obtain a detailed understanding of how CO and O2 bind to Fe in heme proteins, to clarify the topic of FeCO and FeO2 geometries, and to provide probes of E-field/H-bonding interactions. Understanding how ligands bind to metalloproteins; e.g., how O2 binds to and is stabilized by hemoglobin, has considerable long-term health- related implications, since if the important interactions are not known, then improved therapies will be more difficult to engineer.